Lettuce cultivar rio bravo

ABSTRACT

A lettuce cultivar, designated Rio Bravo, is disclosed. The invention relates to the seeds of lettuce cultivar Rio Bravo, to the plants of lettuce cultivar Rio Bravo and to methods for producing a lettuce plant by crossing the cultivar Rio Bravo with itself or another lettuce cultivar. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plants and plant parts produced by those methods. This invention also relates to lettuce cultivars or breeding cultivars and plant parts derived from lettuce cultivar Rio Bravo, to methods for producing other lettuce cultivars, lines or plant parts derived from lettuce cultivar Rio Bravo and to the lettuce plants, varieties, and their parts derived from the use of those methods. The invention further relates to hybrid lettuce seeds, plants, and plant parts produced by crossing cultivar Rio Bravo with another lettuce cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a leaf lettuce (Lactuca sativa L.) variety designated Rio Bravo. All publications cited in this application are herein incorporated by reference.

There are numerous steps in the development of any novel, desirable plant germplasm. Plant breeding begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives. The next step is selection of germplasm that possess the traits to meet the program goals. The goal is to combine in a single variety or hybrid an improved combination of desirable traits from the parental germplasm. These important traits may include increased head size and weight, higher seed yield, improved color, resistance to diseases and insects, tolerance to drought and heat, and better agronomic quality.

Practically speaking, all cultivated forms of lettuce belong to the highly polymorphic species Lactuca sativa that is grown for its edible head and leaves. As a crop, lettuce is grown commercially wherever environmental conditions permit the production of an economically viable yield. Lettuce is the World's most popular salad. In the United States, the principal growing regions are California and Arizona which produce approximately 329,700 acres out of a total annual acreage of more than 333,300 acres (USDA (2005)). Fresh lettuce is available in the United States year-round although the greatest supply is from May through October. For planting purposes, the lettuce season is typically divided into three categories (i.e., early, mid, and late), with the coastal areas planting from January to August, and the desert regions planting from August to December. Fresh lettuce is consumed nearly exclusively as fresh, raw product and occasionally as a cooked vegetable.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae) family. Lettuce is related to chicory, sunflower, aster, dandelion, artichoke, and chrysanthemum. Sativa is one of about 300 species in the genus Lactuca. There are seven different morphological types of lettuce. The crisphead group includes the iceberg and batavian types. Iceberg lettuce has a large, firm head with a crisp texture and a white or creamy yellow interior. The batavian lettuce predates the iceberg type and has a smaller and less firm head. The butterhead group has a small, soft head with an almost oily texture. The romaine, also known as cos lettuce, has elongated upright leaves forming a loose, loaf-shaped head and the outer leaves are usually dark green. Leaf lettuce comes in many varieties, none of which form a head, and include the green oak leaf variety. Latin lettuce looks like a cross between romaine and butterhead. Stem lettuce has long, narrow leaves and thick, edible stems. Oilseed lettuce is a type grown for its large seeds that are pressed to obtain oil. Latin lettuce, stem lettuce, and oilseed lettuce are seldom seen in the United States.

Choice of breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g., F₁ hybrid cultivar, pureline cultivar, etc.). For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants. Popular selection methods commonly include pedigree selection, modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method. Backcross breeding is used to transfer one or a few favorable genes for a highly heritable trait into a desirable cultivar. This approach has been used extensively for breeding disease-resistant cultivars. Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes. The use of recurrent selection in self-pollinating crops depends on the ease of pollination, the frequency of successful hybrids from each pollination, and the number of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation of the efficiency of the breeding procedure. Evaluation criteria vary depending on the goal and objectives, but should include gain from selection per year based on comparisons to an appropriate standard, the overall value of the advanced breeding lines, and the number of successful cultivars produced per unit of input (e.g., per year, per dollar expended, etc.).

Promising advanced breeding lines are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s) for at least three years. The best lines are candidates for new commercial cultivars. Those still deficient in a few traits are used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing and distribution, usually take from ten to twenty years from the time the first cross or selection is made. Therefore, development of new cultivars is a time-consuming process that requires precise forward planning, efficient use of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that are genetically superior, because for most traits the true genotypic value is masked by other confounding plant traits or environmental factors. One method of identifying a superior plant is to observe its performance relative to other experimental plants and to a widely grown standard cultivar. If a single observation is inconclusive, replicated observations provide a better estimate of its genetic worth.

The goal of lettuce plant breeding is to develop new, unique, and superior lettuce cultivars. The breeder initially selects and crosses two or more parental lines, followed by repeated selfing and selection, producing many new genetic combinations. The breeder can theoretically generate billions of different genetic combinations via crossing, selfing, and mutations. The breeder has no direct control at the cellular level. Therefore, two breeders will never develop the same line, or even very similar lines, having the same lettuce traits.

Each year, the plant breeder selects the germplasm to advance to the next generation. This germplasm is grown under different geographical, climatic, and soil conditions, and further selections are then made during, and at the end of, the growing season. The cultivars that are developed are unpredictable. This unpredictability is because the breeder's selection occurs in unique environments, with no control at the DNA level (using conventional breeding procedures), and with millions of different possible genetic combinations being generated. A breeder of ordinary skill in the art cannot predict the final resulting lines he develops, except possibly in a very gross and general fashion. The same breeder cannot produce the same line twice by using the exact same original parents and the same selection techniques. This unpredictability results in the expenditure of large research monies to develop superior lettuce cultivars.

The development of commercial lettuce cultivars requires the development of lettuce varieties, the crossing of these varieties, and the evaluation of the crosses. Pedigree breeding and recurrent selection breeding methods are used to develop cultivars from breeding populations. Breeding programs combine desirable traits from two or more varieties or various broad-based sources into breeding pools from which cultivars are developed by selfing and selection of desired phenotypes. The new cultivars are crossed with other varieties and the hybrids from these crosses are evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement of self-pollinating crops or inbred lines of cross-pollinating crops. Two parents which possess favorable, complementary traits are crossed to produce an F₁. An F₂ population is produced by selfing one or several F₁'s or by intercrossing two F₁'s (sib mating). Selection of the best individuals is usually begun in the F₂ population. Then, beginning in the F₃, the best individuals in the best families are selected. Replicated testing of families, or hybrid combinations involving individuals of these families, often follows in the F₄ generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines or mixtures of phenotypically similar lines are tested for potential release as new cultivars.

Mass and recurrent selections can be used to improve populations of either self- or cross-pollinating crops. A genetically variable population of heterozygous individuals is either identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or line that is the recurrent parent. The source of the trait to be transferred is called the donor parent. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent. After the initial cross, individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.

The single-seed descent procedure in the strict sense refers to planting a segregating population, harvesting a sample of one seed per plant, and using the one-seed sample to plant the next generation. When the population has been advanced from the F₂ to the desired level of inbreeding, the plants from which lines are derived will each trace to different F₂ individuals. The number of plants in a population declines with each generation due to failure of some seeds to germinate or some plants to produce at least one seed. As a result, not all of the F₂ plants originally sampled in the population will be represented by a progeny when generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can also be examined. There are many laboratory-based techniques available for the analysis, comparison and characterization of plant genotype; among these are Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats (SSRs—which are also referred to as Microsatellites), and Single Nucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determine genetic composition. Shoemaker and Olsen (Molecular Linkage Map of Soybean (Glycine max), pp. 6.131-6.138 in S. J. O'Brien (ed.) Genetic Maps: Locus Maps of Complex Genomes, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1993)) developed a molecular genetic linkage map that consisted of 25 linkage groups with about 365 RFLP, 11 RAPD, three classical markers, and four isozyme loci. See also, Shoemaker, R. C., RFLP Map of Soybean, pp. 299-309, in Phillips, R. L. and Vasil, I. K. (eds.), DNA-Based Markers in Plants, Kluwer Academic Press, Dordrecht, the Netherlands (1994).

SSR technology is currently the most efficient and practical marker technology; more marker loci can be routinely used and more alleles per marker locus can be found using SSRs in comparison to RFLPs. For example, Diwan and Cregan described a highly polymorphic microsatellite locus in soybean with as many as 26 alleles. Diwan, N. and Cregan, P. B., Theor. Appl. Genet., 95:22-225 (1997). SNPs may also be used to identify the unique genetic composition of the invention and progeny varieties retaining that unique genetic composition. Various molecular marker techniques may be used in combination to enhance overall resolution.

Molecular markers, which include markers identified through the use of techniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF, SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use of molecular markers is Quantitative Trait Loci (QTL) mapping. QTL mapping is the use of markers which are known to be closely linked to alleles that have measurable effects on a quantitative trait. Selection in the breeding process is based upon the accumulation of markers linked to the positive effecting alleles and/or the elimination of the markers linked to the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for the selection of qualitative traits. For example, markers closely linked to alleles or markers containing sequences within the actual alleles of interest can be used to select plants that contain the alleles of interest during a backcrossing breeding program. The markers can also be used to select toward the genome of the recurrent parent and against the markers of the donor parent. This procedure attempts to minimize the amount of genome from the donor parent that remains in the selected plants. It can also be used to reduce the number of crosses back to the recurrent parent needed in a backcrossing program. The use of molecular markers in the selection process is often called genetic marker enhanced selection or marker-assisted selection. Molecular markers may also be used to identify and exclude certain sources of germplasm as parental varieties or ancestors of a plant by providing a means of tracking genetic profiles through crosses.

Mutation breeding is another method of introducing new traits into lettuce varieties. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased by many different means including temperature, long-term seed storage, tissue culture conditions, radiation (such as X-rays, Gamma rays, neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens (such as base analogs like 5-bromo-uracil), antibiotics, alkylating agents (such as sulfur mustards, nitrogen mustards, epoxides, ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide, hydroxylamine, nitrous acid, or acridines. Once a desired trait is observed through mutagenesis the trait may then be incorporated into existing germplasm by traditional breeding techniques. Details of mutation breeding can be found in Principles of Cultivar Development by Fehr, Macmillan Publishing Company (1993).

The production of double haploids can also be used for the development of homozygous varieties in a breeding program. Double haploids are produced by the doubling of a set of chromosomes from a heterozygous plant to produce a completely homozygous individual. For example, see Wan, et al., Theor. Appl. Genet., 77:889-892 (1989).

Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several reference books (e.g., Principles of Plant Breeding, John Wiley and Son, pp. 115-161 (1960); Allard (1960); Simmonds (1979); Sneep, et al. (1979); Fehr (1987); “Carrots and Related Vegetable Umbelliferae,” Rubatzky, V. E., et al. (1999).

Proper testing should detect any major faults and establish the level of superiority or improvement over current cultivars. In addition to showing superior performance, there must be a demand for a new cultivar that is compatible with industry standards or which creates a new market. The introduction of a new cultivar will incur additional costs to the seed producer, the grower, processor and consumer for special advertising and marketing, altered seed and commercial production practices, and new product utilization. The testing preceding release of a new cultivar should take into consideration research and development costs, as well as technical superiority of the final cultivar. For seed-propagated cultivars, it must be feasible to produce seed easily and economically.

Lettuce in general, and leaf lettuce in particular, is an important and valuable vegetable crop. Thus, a continuing goal of lettuce plant breeders is to develop stable, high yielding lettuce cultivars that are agronomically sound. To accomplish this goal, the lettuce breeder must select and develop lettuce plants with traits that result in superior cultivars.

The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described in conjunction with systems, tools, and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

According to the invention, there is provided a novel lettuce cultivar designated Rio Bravo. This invention thus relates to the seeds of lettuce cultivar Rio Bravo, to the plants of lettuce cultivar Rio Bravo, and to methods for producing a lettuce plant produced by crossing the lettuce cultivar Rio Bravo with itself or another lettuce plant, to methods for producing a lettuce plant containing in its genetic material one or more transgenes, and to the transgenic lettuce plants produced by that method. This invention also relates to methods for producing other lettuce cultivars derived from lettuce cultivar Rio Bravo and to the lettuce cultivar derived by the use of those methods. This invention further relates to hybrid lettuce seeds and plants produced by crossing lettuce cultivar Rio Bravo with another lettuce variety.

In another aspect, the present invention provides regenerable cells for use in tissue culture of lettuce cultivar Rio Bravo. The tissue culture will preferably be capable of regenerating plants having essentially all of the physiological and morphological characteristics of the foregoing lettuce plant, and of regenerating plants having substantially the same genotype as the foregoing lettuce plant. Preferably, the regenerable cells in such tissue cultures will be callus, protoplasts, meristematic cells, cotyledons, hypocotyl, leaves, pollen, embryos, roots, root tips, anthers, pistils, shoots, stems, petiole flowers, and seeds. Still further, the present invention provides lettuce plants regenerated from the tissue cultures of the invention.

Another aspect of the invention is to provide methods for producing other lettuce plants derived from lettuce cultivar Rio Bravo. Lettuce cultivars derived by the use of those methods are also part of the invention.

The invention also relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plant produced by those methods.

In another aspect, the present invention provides for single gene converted plants of Rio Bravo. The single transferred gene may preferably be a dominant or recessive allele. Preferably, the single transferred gene will confer such traits as male sterility, herbicide resistance, insect or pest resistance, modified fatty acid metabolism, modified carbohydrate metabolism, resistance for bacterial, fungal, or viral disease, male fertility, enhanced nutritional quality, and industrial usage. The single gene may be a naturally occurring lettuce gene or a transgene introduced through genetic engineering techniques.

The invention further provides methods for developing lettuce plants in a lettuce plant breeding program using plant breeding techniques including recurrent selection, backcrossing, pedigree breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, and transformation. Seeds, lettuce plants, and parts thereof, produced by such breeding methods are also part of the invention.

In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference by study of the following descriptions.

Definitions

In the description and tables which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a gene, all of which relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedly crosses hybrid progeny back to one of the parents, for example, a first generation hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Big Vein virus. Big vein is a disease of lettuce caused by Lettuce Mirafiori Big Vein Virus which is transmitted by the fungus Olpidium virulentus, with vein clearing and leaf shrinkage resulting in plants of poor quality and reduced marketable value.

Bolting. The premature development of a flowering stalk, and subsequent seed, before a plant produces a food crop. Bolting is typically caused by late planting when temperatures are low enough to cause vernalization of the plants.

Bremia lactucae. An Oomycete that causes downy mildew in lettuce in cooler growing regions.

Core length. Length of the internal lettuce stem measured from the base of the cut and trimmed head to the tip of the stem.

Corky root. A disease caused by the bacterium Sphingomonas suberifaciens, which causes the entire taproot to become brown, severely cracked, and non-functional.

Cotyledon. One of the first leaves of the embryo of a seed plant; typically one or more in monocotyledons, two in dicotyledons, and two or more in gymnosperms.

Essentially all the physiological and morphological characteristics. A plant having essentially all the physiological and morphological characteristics means a plant having the physiological and morphological characteristics of the recurrent parent, except for the characteristics derived from the converted gene.

First water date. The date the seed first receives adequate moisture to germinate. This can and often does equal the planting date.

Gene. As used herein, “gene” refers to a segment of nucleic acid. A gene can be introduced into a genome of a species, whether from a different species or from the same species, using transformation or various breeding methods.

Head diameter. Diameter of the cut and trimmed head, sliced vertically, and measured at the widest point perpendicular to the stem.

Head height. Height of the cut and trimmed head, sliced vertically, and measured from the base of the cut stem to the cap leaf.

Head weight. Weight of saleable lettuce head, cut and trimmed to market specifications.

Lettuce Mosaic virus. A disease that can cause a stunted, deformed, or mottled pattern in young lettuce and yellow, twisted, and deformed leaves in older lettuce.

Maturity date. Maturity refers to the stage when the plants are of full size or optimum weight, in marketable form or shape to be of commercial or economic value.

Nasonovia ribisnigri. A lettuce aphid that colonizes the innermost leaves of the lettuce plant, contaminating areas that cannot be treated easily with insecticides.

Quantitative Trait Loci. Quantitative Trait Loci (QTL) refers to genetic loci that control to some degree, numerically representable traits that are usually continuously distributed.

Ratio of head height/diameter. Head height divided by the head diameter is an indication of the head shape; <1 is flattened, 1=round, and >1 is pointed.

Regeneration. Regeneration refers to the development of a plant from tissue culture.

RHS. RHS refers to the Royal Horticultural Society of England which publishes an official botanical color chart quantitatively identifying colors according to a defined numbering system. The chart may be purchased from Royal Horticulture Society Enterprise Ltd., RHS Garden; Wisley, Woking; Surrey GU236QB, UK.

Single gene converted. Single gene converted or conversion plant refers to plants which are developed by a plant breeding technique called backcrossing or via genetic engineering wherein essentially all of the desired morphological and physiological characteristics of a line are recovered in addition to the single gene transferred into the line via the backcrossing technique or via genetic engineering.

Tip burn. Means a browning of the edges or tips of lettuce leaves that is a physiological response to a lack of calcium.

Tomato Bushy Stunt. A disease which causes stunting of growth, leaf mottling, and deformed or absent fruit.

DETAILED DESCRIPTION OF THE INVENTION

Lettuce cultivar Rio Bravo is a lettuce variety suitable for production in California and the Arizona desert of the United States. Additionally, lettuce cultivar Rio Bravo is highly resistant to Tomato Bushy Stunt Virus (TBSV) and Downy Mildew. It is the result of numerous generations of plant selections chosen for its type and disease resistances.

The cultivar has shown uniformity and stability for the traits, within the limits of environmental influence for the traits. It has been self-pollinated a sufficient number of generations with careful attention to uniformity of plant type. The line has been increased with continued observation for uniformity. No variant traits have been observed or are expected in cultivar Rio Bravo.

Lettuce cultivar Rio Bravo has the following morphologic and other characteristics, described in Table 1.

TABLE 1 VARIETY DESCRIPTION INFORMATION Plant: Type: Green Romaine Maturity date: 65 days (Summer), 115 days (winter) from first water date. Seed: Color: Black Light dormancy: Light not required Heat dormancy: Susceptible Cotyledon (to fourth leaf stage): Shape: Broad Undulation: Flat Anthocyanin distribution: Absent Rolling: Absent Cupping: Uncupped Reflexing: None Mature Leaves: Margin: Incision depth: Absent/Shallow Indentation: Shallowly Dentate Undulation of the apical margin: Absent/Slight Hue of green color of outer leaves: Dark Green Anthocyanin distribution: Absent Glossiness: Dull Blistering: Moderate Thickness: Thick Trichomes: Absent Plant at Market Stage Head shape: Slight V Shaped Head size class: Large Head weight (g): 720.7 Head firmness: Moderate Core: Diameter at base of head (cm):  3.51 Core height from base of head  6.44 to apex (cm): Primary Regions of Adaptation: Spring area: Salinas, California, Imperial, California, San Joaquin, California, and Yuma, Arizona (United States) Summer area: Salinas, California, Santa Maria, California, and San Benito, California (United States) Autumn area: Yuma, Arizona, Imperial, California, and Salinas, California (United States) Winter area: Yuma, Arizona, Imperial, California, and Coachella, California (United States) Disease and Stress Reactions: Tomato Bushy Stunt Virus Highly resistant (TBSV): Big Vein: Intermediate Downy Mildew (Bremia lactucae): Highly resistant Tipburn: Tolerant Heat: Intermediate Cold: Tolerant Brown Rib: Resistant Pink Rib: Resistant Rusty Brown Discoloration: Resistant Internal Rib Necrosis (Blackheart, Resistant Gray Rib, Gray Streak):

Additional methods include, but are not limited to, expression vectors introduced into plant tissues using a direct gene transfer method, such as microprojectile-mediated delivery, DNA injection, electroporation, and the like. More preferably, expression vectors are introduced into plant tissues by using either microprojectile-mediated delivery with a biolistic device or by using Agrobacterium-mediated transformation. Transformed plants obtained with the protoplasm of the invention are intended to be within the scope of this invention.

Further Embodiments of the Invention

With the advent of molecular biological techniques that have allowed the isolation and characterization of genes that encode specific protein products, scientists in the field of plant biology developed a strong interest in engineering the genome of plants to contain and express foreign genes, or additional, or modified versions of native, or endogenous, genes (perhaps driven by different promoters) in order to alter the traits of a plant in a specific manner. Any DNA sequences, whether from a different species or from the same species, which are introduced into the genome using transformation or various breeding methods, are referred to herein collectively as “transgenes.” Over the last fifteen to twenty years, several methods for producing transgenic plants have been developed, and the present invention, in particular embodiments, also relates to transformed versions of the claimed line.

Nucleic acids or polynucleotides refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. These terms also encompass untranslated sequence located at both the 3′ and 5′ ends of the coding region of the gene: at least about 1000 nucleotides of sequence upstream from the 5′ end of the coding region and at least about 200 nucleotides of sequence downstream from the 3′ end of the coding region of the gene. Less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2′-hydroxy in the ribose sugar group of the RNA can also be made. The antisense polynucleotides and ribozymes can consist entirely of ribonucleotides, or can contain mixed ribonucleotides and deoxyribonucleotides. The polynucleotides of the invention may be produced by any means, including genomic preparations, cDNA preparations, in vitro synthesis, RT-PCR, and in vitro or in vivo transcription.

Plant transformation involves the construction of an expression vector that will function in plant cells. Such a vector comprises DNA comprising a gene under control of, or operatively linked to, a regulatory element (for example, a promoter). The expression vector may contain one or more such operably linked gene/regulatory element combinations. The vector(s) may be in the form of a plasmid, and can be used alone or in combination with other plasmids, to provide transformed lettuce plants using transformation methods as described below to incorporate transgenes into the genetic material of the lettuce plant(s).

Expression Vectors for Lettuce Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linked to a regulatory element (for example, a promoter) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker. Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII) gene, isolated from transposon Tn5, which when placed under the control of plant regulatory signals confers resistance to kanamycin. Fraley, et al., PNAS, 80:4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase, the bleomycin resistance determinant. Hayford, et al., Plant Physiol., 86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86 (1987); Svab, et al., Plant Mol. Biol., 14:197 (1990); Hille, et al., Plant Mol. Biol., 7:171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate, or bromoxynil. Comai, et al., Nature, 317:741-744 (1985); Gordon-Kamm, et al., Plant Cell, 2:603-618 (1990); and Stalker, et al., Science, 242:419-423 (1988).

Selectable marker genes for plant transformation that are not of bacterial origin include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactate synthase. Eichholtz, et al., Somatic Cell Mol. Genet., 13:67 (1987); Shah, et al., Science, 233:478 (1986); and Charest, et al., Plant Cell Rep., 8:643 (1990).

Another class of marker genes for plant transformation requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include α-glucuronidase (GUS), α-galactosidase, luciferase and chloramphenicol, acetyltransferase. Jefferson, R. A., Plant Mol. Biol., 5:387 (1987); Teeri, et al., EMBO J., 8:343 (1989); Koncz, et al., PNAS, 84:131 (1987); and DeBlock, et al., EMBO J., 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not require destruction of plant tissues are available. Molecular Probes, Publication 2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J. Cell Biol., 115:151a (1991). However, these in vivo methods for visualizing GUS activity have not proven useful for recovery of transformed cells because of low sensitivity, high fluorescent backgrounds, and limitations associated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells. Chalfie, et al., Science, 263:802 (1994). GFP and mutants of GFP may be used as screenable markers.

Expression Vectors for Lettuce Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotide sequence comprising a regulatory element (for example, a promoter). Several types of promoters are now well known in the transformation arts, as are other regulatory elements that can be used alone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue-preferred.” Promoters which initiate transcription only in certain tissue are referred to as “tissue-specific.” A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most environmental conditions.

A. Inducible Promoters:

An inducible promoter is operably linked to a gene for expression in lettuce. Optionally, the inducible promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce. With an inducible promoter, the rate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward, et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary inducible promoters include, but are not limited to, that from the ACEI system which responds to copper (Meft, et al., PNAS, 90:4567-4571 (1993)); In2 gene from maize which responds to benzenesulfonamide herbicide safeners (Hershey, et al., Mol. Gen. Genet., 227:229-237 (1991) and Gatz, et al., Mol. Gen. Genet., 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz, et al., Mol. Gen. Genet., 227:229-237 (1991)). A particularly preferred inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone. Schena, et al., PNAS, 88:0421 (1991).

B. Constitutive Promoters:

A constitutive promoter is operably linked to a gene for expression in lettuce or the constitutive promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce.

Many different constitutive promoters can be utilized in the instant invention. Exemplary constitutive promoters include, but are not limited to, the promoters from plant viruses such as the 35S promoter from CaMV (Odell, et al., Nature, 313:810-812 (1985)) and the promoters from such genes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990)); ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) and Christensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last, et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al., EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol. Gen. Genet., 231:276-285 (1992) and Atanassova, et al., Plant J., 2 (3):291-300 (1992)). The ALS promoter, Xba1/Nco1 fragment 5′ to the Brassica napus ALS3 structural gene (or a nucleotide sequence similarity to said Xba1/Nco1 fragment), represents a particularly useful constitutive promoter. See PCT Application No. WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters:

A tissue-specific promoter is operably linked to a gene for expression in lettuce. Optionally, the tissue-specific promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce. Plants transformed with a gene of interest operably linked to a tissue-specific promoter produce the protein product of the transgene exclusively, or preferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in the instant invention. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to, a root-preferred promoter, such as that from the phaseolin gene (Murai, et al., Science, 23:476-482 (1983) and Sengupta-Gopalan, et al., PNAS, 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such as that from cab or rubisco (Simpson, et al., EMBO J., 4(11):2723-2729 (1985) and Timko, et al., Nature, 318:579-582 (1985)); an anther-specific promoter such as that from LAT52 (Twell, et al., Mol. Gen. Genet., 217:240-245 (1989)); a pollen-specific promoter such as that from Zm13 (Guerrero, et al., Mol. Gen. Genet., 244:161-168 (1993)) or a microspore-preferred promoter such as that from apg (Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartment such as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, or mitochondrion, or for secretion into the apoplast, is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the 5′ and/or 3′ region of a gene encoding the protein of interest. Targeting sequences at the 5′ and/or 3′ end of the structural gene may determine, during protein synthesis and processing, where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either an intracellular organelle or subcellular compartment or for secretion to the apoplast. Many signal sequences are known in the art. See, for example, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S., Master's Thesis, Iowa State University (1993); Knox, C., et al., “Structure and Organization of Two Divergent Alpha-Amylase Genes from Barley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., Plant Physiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496 (1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell. Biol., 108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991); Kalderon, et al., A short amino acid sequence able to specify nuclear location, Cell, 39:499-509 (1984); and Steifel, et al., Expression of a maize cell wall hydroxyproline-rich glycoprotein gene in early leaf and root vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreign protein can be produced in commercial quantities. Thus, techniques for the selection and propagation of transformed plants, which are well understood in the art, yield a plurality of transgenic plants which are harvested in a conventional manner, and a foreign protein then can be extracted from a tissue of interest or from total biomass. Protein extraction from plant biomass can be accomplished by known methods which are discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6 (1981).

According to a preferred embodiment, the transgenic plant provided for commercial production of foreign protein is lettuce. In another preferred embodiment, the biomass of interest is seed. For the relatively small number of transgenic plants that show higher levels of expression, a genetic map can be generated, primarily via conventional RFLP, PCR, and SSR analysis, which identifies the approximate chromosomal location of the integrated DNA molecule. For exemplary methodologies in this regard, see Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson Eds., 269:284, CRC Press, Boca Raton (1993). Map information concerning chromosomal location is useful for proprietary protection of a subject transgenic plant. If unauthorized propagation is undertaken and crosses made with other germplasm, the map of the integration region can be compared to similar maps for suspect plants, to determine if the latter have a common parentage with the subject plant. Map comparisons would involve hybridizations, RFLP, PCR, SSR, and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can be expressed in transformed plants. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes implicated in this regard include, but are not limited to, those categorized below:

A. Genes That Confer Resistance to Pests or Disease and That Encode:

1. Plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant line can be transformed with a cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example, Jones, et al., Science, 266:789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin, et al., Science, 262:1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); and Mindrinos, et al., Cell, 78:1089 (1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae).

2. A Bacillus thuringiensis protein, a derivative thereof, or a synthetic polypeptide modeled thereon. See, for example, Geiser, et al., Gene, 48:109 (1986), who disclose the cloning and nucleotide sequence of a Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes can be purchased from American Type Culture Collection, Manassas, Va., for example, under ATCC Accession Nos. 40098, 67136, 31995, and 31998.

3. A lectin. See, for example, the disclosure by Van Damme, et al., Plant Mol. Biol., 24:25 (1994), who disclose the nucleotide sequences of several Clivia miniata mannose-binding lectin genes.

4. A vitamin-binding protein such as avidin. See PCT Application No. US 93/06487, the contents of which are hereby incorporated by reference. The application teaches the use of avidin and avidin homologues as larvicides against insect pests.

5. An enzyme inhibitor, for example, a protease or proteinase inhibitor, or an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem., 262:16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor); Huub, et al., Plant Mol. Biol., 21:985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I); and Sumitani, et al., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus α-amylase inhibitor).

6. An insect-specific hormone or pheromone, such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock, et al., Nature, 344:458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.

7. An insect-specific peptide or neuropeptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloning yields DNA coding for insect diuretic hormone receptor) and Pratt, et al., Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin is identified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 to Tomalski, et al., who disclose genes encoding insect-specific, paralytic neurotoxins.

8. An insect-specific venom produced in nature, by a snake, a wasp, etc. For example, see Pang, et al., Gene, 116:165 (1992), for disclosure of heterologous expression in plants of a gene coding for a scorpion insectotoxic peptide.

9. An enzyme responsible for a hyper-accumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative, or another non-protein molecule with insecticidal activity.

10. An enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase, and a glucanase, whether natural or synthetic. See PCT Application No. WO 93/02197 in the name of Scott, et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC under Accession Nos. 39637 and 67152. See also, Kramer, et al., Insect Biochem. Mol. Biol., 23:691 (1993), who teach the nucleotide sequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck, et al., Plant Mol. Biol., 21:673 (1993), who provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene.

11. A molecule that stimulates signal transduction. For example, see the disclosure by Botella, et al., Plant Mol. Biol., 24:757 (1994), of nucleotide sequences for mung bean calmodulin cDNA clones, and Griess, et al., Plant Physiol., 104:1467 (1994), who provide the nucleotide sequence of a maize calmodulin cDNA clone.

12. A hydrophobic moment peptide. See PCT Application No. WO 95/16776 (disclosure of peptide derivatives of tachyplesin which inhibit fungal plant pathogens) and PCT Application No. WO 95/18855 (teaches synthetic antimicrobial peptides that confer disease resistance), the respective contents of which are hereby incorporated by reference.

13. A membrane permease, a channel former, or a channel blocker. For example, see the disclosure of Jaynes, et al., Plant Sci., 89:43 (1993), of heterologous expression of a cecropin-β, lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum.

14. A viral-invasive protein or a complex toxin derived therefrom. For example, the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See Beachy, et al., Ann. Rev. Phytopathol., 28:451 (1990). Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus, and tobacco mosaic virus. Id.

15. An insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. See Taylor, et al., Abstract #497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions, Edinburgh, Scotland (1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).

16. A virus-specific antibody. See, for example, Tavladoraki, et al., Nature, 366:469 (1993), who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.

17. A developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitate fungal colonization and plant nutrient released by solubilizing plant cell wall homo-α-1,4-D-galacturonase. See Lamb, et al., Bio/technology, 10:1436 (1992). The cloning and characterization of a gene which encodes a bean endopolygalacturonase-inhibiting protein is described by Toubart, et al., Plant J, 2:367 (1992).

18. A developmental-arrestive protein produced in nature by a plant. For example, Logemann, et al., Bio/technology, 10:305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.

19. A lettuce mosaic potyvirus (LMV) coat protein gene introduced into Lactuca sativa in order to increase its resistance to LMV infection. See Dinant, et al., Mol. Breeding, 3:1, 75-86 (1997).

Any of the above listed disease or pest resistance genes (1-19) can be introduced into the claimed lettuce cultivar through a variety of means including but not limited to transformation and crossing.

B. Genes That Confer Resistance to an Herbicide:

1. An herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee, et al., EMBO J., 7:1241 (1988) and Miki, et al., Theor. Appl. Genet., 80:449 (1990), respectively.

2. Glyphosate (resistance conferred by mutant 5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes, respectively) and other phosphono compounds, such as glufosinate (phosphinothricin acetyl transferase (PAT), dicamba and Streptomyces hygroscopicus phosphinothricin-acetyl transferase PAT bar genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSPS which can confer glyphosate resistance. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC Accession No. 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. See also, Umaballava-Mobapathie in Transgenic Research, 8:1, 33-44 (1999) that discloses Lactuca sativa resistant to glufosinate. European Patent Application No. 0 333 033 to Kumada, et al., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides, such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in European Application No. 0 242 246 to Leemans, et al. DeGreet et al., Bio/technology, 7:61 (1989), describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. Exemplary of genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2, and Acc1-S3 genes described by Marshall, et al., Theor. Appl. Genet., 83:435 (1992).

3. An herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al., Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes, et al., Biochem. J., 285:173 (1992).

4. Acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants. See Hattori, et al., Mol. Gen. Genet., 246:419 (1995). Other genes that confer tolerance to herbicides include a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., Plant Physiol., 106:17 (1994)), genes for glutathione reductase and superoxide dismutase (Aono, et al., Plant Cell Physiol., 36:1687 (1995)), and genes for various phosphotransferases (Datta, et al., Plant Mol. Biol., 20:619 (1992)).

5. Protoporphyrinogen oxidase (protox) is necessary for the production of chlorophyll, which is necessary for all plant survival. The protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837, 5,767,373, and International Publication WO 01/12825.

Any of the above listed herbicide genes (1-5) can be introduced into the claimed lettuce cultivar through a variety of means including, but not limited to, transformation and crossing.

C. Genes that Confer or Contribute to a Value-Added Trait, Such as:

1. Increased iron content of the lettuce, for example, by introducing into a plant a soybean ferritin gene as described in Goto, et al., Acta Horticulturae., 521, 101-109 (2000).

2. Decreased nitrate content of leaves, for example, by introducing into a lettuce a gene coding for a nitrate reductase. See, for example, Curtis, et al., Plant Cell Rep., 18:11, 889-896 (1999).

3. Increased sweetness of the lettuce by introducing a gene coding for monellin that elicits a flavor 100,000 times sweeter than sugar on a molar basis. See Penarrubia, et al., Bio/technology, 10:561-564 (1992).

4. Modified fatty acid metabolism, for example, by introducing into a plant an antisense gene of stearyl-ACP desaturase to increase stearic acid content of the plant. See Knultzon, et al., PNAS, 89:2625 (1992).

5. Modified carbohydrate composition effected, for example, by introducing into plants a gene coding for an enzyme that alters the branching pattern of starch. See Shiroza, et al., J. Bacteriol., 170:810 (1988) (nucleotide sequence of Streptococcus mutants fructosyltransferase gene); Steinmetz, et al., Mol. Gen. Genet., 20:220 (1985) (nucleotide sequence of Bacillus subtilis levansucrase gene); Pen, et al., Bio/technology, 10:292 (1992) (production of transgenic plants that express Bacillus lichenifonnis α-amylase); Elliot, et al., Plant Mol. Biol., 21:515 (1993) (nucleotide sequences of tomato invertase genes); Søgaard, et al., J. Biol. Chem., 268:22480 (1993) (site-directed mutagenesis of barley α-amylase gene); and Fisher, et al., Plant Physiol., 102:1045 (1993) (maize endosperm starch branching enzyme II).

D. Genes that Control Male-Sterility:

1. Introduction of a deacetylase gene under the control of a tapetum-specific promoter and with the application of the chemical N—Ac-PPT. See International Publication WO 01/29237.

2. Introduction of various stamen-specific promoters. See International Publications WO 92/13956 and WO 92/13957.

3. Introduction of the barnase and the barstar genes. See Paul, et al., Plant Mol. Biol., 19:611-622 (1992).

Methods for Lettuce Transformation

Numerous methods for plant transformation have been developed, including biological and physical, plant transformation protocols. See, for example, Miki, et al., “Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber, et al., “Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick and Thompson Eds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation:

One method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch, et al., Science, 227:1229 (1985); Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449 (1994); Torres, et al., Plant Cell Tissue and Organ Culture, 34:3, 279-285 (1993); and Dinant, et al., Molecular Breeding, 3:1, 75-86 (1997). A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I., Crit. Rev. Plant Sci., 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber, et al., supra, Miki, et al., supra, and Moloney, et al., Plant Cell Rep., 8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer:

Several methods of plant transformation collectively referred to as direct gene transfer have been developed as an alternative to Agrobacterium-mediated transformation. A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 μm to 4 μm. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 m/s to 600 m/s which is sufficient to penetrate plant cell walls and membranes. Russell, D. R., et al., Plant Cell Rep., 12 (3, Jan.), 165-169 (1993); Aragao, F. J. L., et al., Plant Mol. Biol., 20 (2, October), 357-359 (1992); Aragao, F. J. L., et al., Plant Cell Rep., 12 (9, July), 483-490 (1993); Aragao, Theor. Appl. Genet., 93:142-150 (1996); Kim, J., Minamikawa, T., Plant Sci., 117:131-138 (1996); Sanford, et al., Part. Sci. Technol., 5:27 (1987); Sanford, J. C., Trends Biotech., 6:299 (1988); Klein, et al., Bio/technology, 6:559-563 (1988); Sanford, J. C., Physiol. Plant, 7:206 (1990); Klein, et al., Bio/technology, 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication of target cells. Zhang, et al., Bio/technology, 9:996 (1991). Alternatively, liposome and spheroplast fusion have been used to introduce expression vectors into plants. Deshayes, et al., EMBO J., 4:2731 (1985) and Christou, et al., PNAS, 84:3962 (1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol, or poly-L-ornithine has also been reported. Hain, et al., Mol. Gen. Genet., 199:161 (1985) and Draper, et al., Plant Cell Physiol., 23:451 (1982). Electroporation of protoplasts and whole cells and tissues have also been described. Saker, M., Kuhne, T., Biologia Plantarum, 40(4):507-514 (1997/98); Donn, et al., In Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin, et al., Plant Cell, 4:1495-1505 (1992); and Spencer, et al., Plant Mol. Biol., 24:51-61 (1994). See also Chupean, et al., Bio/technology, 7:5, 503-508 (1989).

Following transformation of lettuce target tissues, expression of the above-described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used for producing a transgenic line. The transgenic line could then be crossed with another (non-transformed or transformed) line in order to produce a new transgenic lettuce line. Alternatively, a genetic trait which has been engineered into a particular lettuce cultivar using the foregoing transformation techniques could be introduced into another line using traditional backcrossing techniques that are well known in the plant breeding arts. For example, a backcrossing approach could be used to move an engineered trait from a public, non-elite inbred line into an elite inbred line, or from an inbred line containing a foreign gene in its genome into an inbred line or lines which do not contain that gene. As used herein, “crossing” can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.

Gene Conversions

When the term “lettuce plant” is used in the context of the present invention, this also includes any gene conversions of that variety. The term “gene converted plant” as used herein refers to those lettuce plants which are developed by backcrossing, genetic engineering, or mutation, wherein essentially all of the desired morphological and physiological characteristics of a variety are recovered in addition to the one or more genes transferred into the variety via the backcrossing technique, genetic engineering, or mutation. Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the variety. The term “backcrossing” as used herein refers to the repeated crossing of a hybrid progeny back to the recurrent parent, i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times to the recurrent parent. The parental lettuce plant which contributes the gene for the desired characteristic is termed the “nonrecurrent” or “donor parent.” This terminology refers to the fact that the nonrecurrent parent is used one time in the backcross protocol and therefore does not recur. The parental lettuce plant to which the gene or genes from the nonrecurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol. Poehlman & Sleper (1994) and Fehr (1993). In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (nonrecurrent parent) that carries the gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a lettuce plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the transferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for a successful backcrossing procedure. The goal of a backcross protocol is to alter or substitute a trait or characteristic in the original line. To accomplish this, a gene of the recurrent cultivar is modified or substituted with the desired gene from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, and therefore the desired physiological and morphological, constitution of the original line. The choice of the particular nonrecurrent parent will depend on the purpose of the backcross. One of the major purposes is to add some commercially desirable, agronomically important trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered to determine an appropriate testing protocol. Although backcrossing methods are simplified when the characteristic being transferred is a dominant allele, a recessive allele may also be transferred. In this instance it may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.

Many gene traits have been identified that are not regularly selected in the development of a new line but that can be improved by backcrossing techniques. Gene traits may or may not be transgenic. Examples of these traits include, but are not limited to, male sterility, modified fatty acid metabolism, modified carbohydrate metabolism, herbicide resistance, resistance for bacterial, fungal, or viral disease, insect resistance, enhanced nutritional quality, industrial usage, yield stability, and yield enhancement. These genes are generally inherited through the nucleus. Several of these gene traits are described in U.S. Pat. Nos. 5,777,196, 5,948,957, and 5,969,212, the disclosures of which are specifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture and regeneration. Tissue culture of various tissues of lettuce and regeneration of plants therefrom is well known and widely published. For example, reference may be had to Teng, et al., HortScience, 27:9, 1030-1032 (1992); Teng, et al., HortScience, 28:6, 669-1671 (1993); Zhang, et al., Journal of Genetics and Breeding, 46:3, 287-290 (1992); Webb, et al., Plant Cell Tissue and Organ Culture, 38:1, 77-79 (1994); Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449 (1994); Nagata, et al., Journal for the American Society for Horticultural Science, 125:6, 669-672 (2000); and Ibrahim, et al., Plant Cell Tissue and Organ Culture, 28(2), 139-145 (1992). It is clear from the literature that the state of the art is such that these methods of obtaining plants are routinely used and have a very high rate of success. Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce lettuce plants having the physiological and morphological characteristics of variety Rio Bravo.

As used herein, the term “tissue culture” indicates a composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant. Exemplary types of tissue cultures are protoplasts, calli, meristematic cells, and plant cells that can generate tissue culture that are intact in plants or parts of plants, such as leaves, pollen, embryos, roots, root tips, anthers, pistils, flowers, seeds, petioles, suckers, and the like. Means for preparing and maintaining plant tissue culture are well known in the art. By way of example, a tissue culture comprising organs has been used to produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and 5,977,445 describe certain techniques, the disclosures of which are incorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a lettuce plant by crossing a first parent lettuce plant with a second parent lettuce plant wherein the first or second parent lettuce plant is a lettuce plant of cultivar Rio Bravo. Further, both first and second parent lettuce plants can come from lettuce cultivar Rio Bravo. Thus, any such methods using lettuce cultivar Rio Bravo are part of this invention: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using lettuce cultivar Rio Bravo as at least one parent are within the scope of this invention, including those developed from cultivars derived from lettuce cultivar Rio Bravo. Advantageously, this lettuce cultivar could be used in crosses with other, different, lettuce plants to produce the first generation (F₁) lettuce hybrid seeds and plants with superior characteristics. The cultivar of the invention can also be used for transformation where exogenous genes are introduced and expressed by the cultivar of the invention. Genetic variants created either through traditional breeding methods using lettuce cultivar Rio Bravo or through transformation of cultivar Rio Bravo by any of a number of protocols known to those of skill in the art are intended to be within the scope of this invention.

The following describes breeding methods that may be used with lettuce cultivar Rio Bravo in the development of further lettuce plants. One such embodiment is a method for developing cultivar Rio Bravo progeny lettuce plants in a lettuce plant breeding program comprising: obtaining the lettuce plant, or a part thereof, of cultivar Rio Bravo, utilizing said plant or plant part as a source of breeding material, and selecting a lettuce cultivar Rio Bravo progeny plant with molecular markers in common with cultivar Rio Bravo and/or with morphological and/or physiological characteristics selected from the characteristics listed in Table 1. Breeding steps that may be used in the lettuce plant breeding program include pedigree breeding, backcrossing, mutation breeding, and recurrent selection. In conjunction with these steps, techniques such as RFLP-enhanced selection, genetic marker enhanced selection (for example, SSR markers), and the making of double haploids may be utilized.

Another method involves producing a population of lettuce cultivar Rio Bravo progeny lettuce plants, comprising crossing cultivar Rio Bravo with another lettuce plant, thereby producing a population of lettuce plants, which, on average, derive 50% of their alleles from lettuce cultivar Rio Bravo. A plant of this population may be selected and repeatedly selfed or sibbed with a lettuce cultivar resulting from these successive filial generations. One embodiment of this invention is the lettuce cultivar produced by this method and that has obtained at least 50% of its alleles from lettuce cultivar Rio Bravo.

One of ordinary skill in the art of plant breeding would know how to evaluate the traits of two plant varieties to determine if there is no significant difference between the two traits expressed by those varieties. For example, see Fehr and Walt, Principles of Cultivar Development, pp. 261-286 (1987). Thus the invention includes lettuce cultivar Rio Bravo progeny lettuce plants comprising a combination of at least two cultivar Rio Bravo traits selected from the group consisting of those listed in Table 1 or the cultivar Rio Bravo combination of traits listed in the Summary of the Invention, so that said progeny lettuce plant is not significantly different for said traits than lettuce cultivar Rio Bravo as determined at the 5% significance level when grown in the same environmental conditions. Using techniques described herein, molecular markers may be used to identify said progeny plant as a lettuce cultivar Rio Bravo progeny plant. Mean trait values may be used to determine whether trait differences are significant, and preferably the traits are measured on plants grown under the same environmental conditions. Once such a variety is developed, its value is substantial since it is important to advance the germplasm base as a whole in order to maintain or improve traits such as yield, disease resistance, pest resistance, and plant performance in extreme environmental conditions.

Progeny of lettuce cultivar Rio Bravo may also be characterized through their filial relationship with lettuce cultivar Rio Bravo, as for example, being within a certain number of breeding crosses of lettuce cultivar Rio Bravo. A breeding cross is a cross made to introduce new genetics into the progeny, and is distinguished from a cross, such as a self or a sib cross, made to select among existing genetic alleles. The lower the number of breeding crosses in the pedigree, the closer the relationship between lettuce cultivar Rio Bravo and its progeny. For example, progeny produced by the methods described herein may be within 1, 2, 3, 4, or 5 breeding crosses of lettuce cultivar Rio Bravo.

As used herein, the term “plant” includes plant cells, plant protoplasts, plant cell tissue cultures from which lettuce plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants, such as leaves, pollen, embryos, cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers, ovules, seeds, stems, and the like.

Tables

Table 2 compares the length of the cotyledon leaf in millimeters of 20 day old seedlings of leaf lettuce cultivar Rio Bravo with commercial lettuce cultivars Green Thunder and True Heart. Data were taken in 2010 in SALINAS, CALIFORNIA on 20 plants of each variety. Column 1 shows the cotyledon length for Rio Bravo, column 2 shows the cotyledon length for Green Thunder, and column 3 shows the cotyledon length for True Heart.

TABLE 2 Rio Bravo Green Thunder True Heart 21 18 19 17 20 17 19 21 20 19 20 18 19 21 21 20 20 18 18 19 19 18 18 20 19 22 20 18 20 19 18 21 22 19 20 22 20 20 18 21 20 18 18 21 16 20 15 18 18 22 19 21 18 18 18 20 18 17 20 19 Average 18.9 19.8 18.95

Table 3 compares the width of the cotyledon leaf in millimeters of 20 day old seedlings of leaf lettuce cultivar Rio Bravo with commercial lettuce cultivars Green Thunder and True Heart. Data were taken in 2010 in SALINAS, CALIFORNIA on 20 plants of each variety. Column 1 lists the cotyledon width for Rio Bravo, column 2 lists the cotyledon width for Green Thunder, and column 3 lists the cotyledon width for True Heart.

TABLE 3 Rio Bravo Green Thunder True Heart 10 10 9 10 10 9 10 10 10 10 10 9 10 10 11 9 9 9 10 9 9 10 9 9 8 11 11 9 9 10 10 11 10 11 9 11 10 10 9 10 9 10 10 10 8 8 8 10 10 10 10 10 9 9 9 10 9 9 10 9 Average 9.65 9.65 9.55

Table 4 compares the cotyledon leaf index (calculated by dividing the cotyledon leaf length by the cotyledon leaf width) in 20 day old seedlings of leaf lettuce cultivar Rio Bravo with commercial lettuce cultivars Green Thunder and True Heart. Data were taken in 2010 in SALINAS, CALIFORNIA on 20 plants of each variety. Column 1 lists the cotyledon index for Rio Bravo, column 2 lists the cotyledon index for Green Thunder, and column 3 lists the cotyledon index for True Heart.

TABLE 4 Rio Bravo Green Thunder True Heart 2.1 1.8 2.1 1.7 2.0 1.9 1.9 2.1 2.0 1.9 2.0 2.0 1.9 2.1 1.9 2.2 2.2 2.0 1.8 2.1 2.1 1.8 2.0 2.2 2.4 2.0 1.8 2.0 2.2 1.9 1.8 1.9 2.2 1.7 2.2 2.0 2.0 2.0 2.0 2.1 2.2 1.8 1.8 2.1 2.0 2.5 1.9 1.8 1.8 2.2 1.9 2.1 2.0 2.0 2.0 2.0 2.0 1.9 2.0 2.1 Average 1.9707 2.0542 1.9886

Table 5 compares the length of the 4^(th) true leaf measured in centimeters of 20 day old seedlings of leaf lettuce cultivar Rio Bravo with commercial lettuce cultivars Green Thunder and True Heart. Data were taken in 2010 in SALINAS, CALIFORNIA on 20 plants of each variety. Column 1 shows the length of the 4^(th) true leaf for Rio Bravo, column 2 shows the length of the 4^(th) true leaf for Green Thunder, and column 3 shows the length of the 4^(th) true leaf for True Heart. The sum, average, and variance of the measurements for each variety is shown in the last three rows.

TABLE 5 Rio Green Bravo Thunder True Heart 4.8 5.3 4.4 4.2 4.5 5.2 3.4 4.5 5.0 3.8 3.8 5.2 5.2 4.7 5.4 6.3 4.2 4.5 5.5 5.0 5.6 4.8 5.5 5.2 6.5 4.5 4.5 5.5 4.5 5.2 5.5 4.2 4.8 5.8 4.0 4.2 5.6 3.9 5.2 6.2 4.3 5.5 5.0 4.5 4.6 4.0 5.0 3.8 5.5 3.7 5.2 5.4 3.8 4.2 4.4 3.2 5.0 5.0 4.6 3.2 Sum 102.4 87.7 95.9 Average 5.12 4.385 4.495 Variance 0.7038 0.3192 0.3784

Table 6 shows the results of a single-factor ANOVA testing for the length of the 4^(th) true leaf between varieties, and within the varieties, Rio Bravo, Green Thunder, and True Heart from data in Table 5. Column 1 shows the source of variation, column 2 shows the sum of squares, column 3 shows the degrees of freedom, column 4 shows the mean square, column 5 shows the F-value, column 6 shows the P-value, and column 7 shows the F critical value. ANOVA results indicate a significant difference in the length of the 4^(th) true leaf between the varieties at 20 days old.

TABLE 6 Source of Sum of Degrees of Mean Variation Squares Freedom Square F-value P-value F crit Between 5.4263 2 2.7132 5.8080 0.0051 3.1588 Varieties Within 26.627 57 0.4671 Varieties Total 32.053 59

Table 7 compares the width of the 4^(th) true leaf measured in millimeters of 20 day old seedlings of leaf lettuce cultivar Rio Bravo with commercial lettuce cultivars Green Thunder and True Heart. Data were taken in 2010 in SALINAS, CALIFORNIA on 20 plants of each variety. Column 1 shows the width of the 4^(th) true leaf for Rio Bravo, column 2 shows the width of the 4^(th) true leaf for Green Thunder, and column 3 shows the width of the 4^(th) true leaf for True Heart. The sum, average, and variance of the measurements for each variety is shown in the last three rows.

TABLE 7 Rio Green Bravo Thunder True Heart 2.4 2.5 2.2 2.4 2.2 2.8 1.8 2.5 2.6 2.2 2.0 2.6 2.8 2.5 2.6 3.2 2.2 2.3 2.8 2.7 2.6 2.6 2.8 2.6 3.2 2.5 2.2 2.7 2.4 2.8 3.0 2.1 2.3 3.1 2.0 2.2 3.0 2.0 2.8 3.0 2.2 2.2 2.4 2.0 2.4 2.1 2.5 2.0 3.0 1.8 2.8 3.0 1.9 2.2 2.2 1.8 2.4 2.6 2.1 1.6 Sum 53.5 44.7 48.5 Average 2.6750 2.2350 2.4100 Variance 0.1620 0.0877 0.0978

Table 8 shows the results of a single-factor ANOVA testing for the width of the 4^(th) true leaf between varieties, and within the varieties, Rio Bravo, Green Thunder, and True Heart from data in Table 7. Column 1 shows the source of variation, column 2 shows the sum of squares, column 3 shows the degrees of freedom, column 4 shows the mean square, column 5 shows the F-value, column 6 shows the P-value, and column 7 shows the F critical value. ANOVA results indicate a significant difference in the width of the 4^(th) true leaf between the varieties at 20 days old.

TABLE 8 Source of Sum of Degrees of Mean Variation Squares Freedom Square F-value P-value F crit Between 1.963 2 0.9815 8.4753 0.0006 3.1588 Varieties Within 6.601 57 0.1158 Varieties Total 8.564 59

Table 9 compares the 4^(th) leaf index (calculated by dividing the 4^(th) leaf length by the 4^(th) leaf width) of 20 day old seedlings of leaf lettuce cultivar Rio Bravo with commercial lettuce cultivars Green Thunder and True Heart. Data were taken in 2010 in SALINAS, CALIFORNIA on 20 plants of each variety. Column 1 shows the 4^(th) leaf index for Rio Bravo, column 2 shows the 4^(th) leaf index for Green Thunder, and column 3 shows the 4^(th) leaf index for True Heart.

TABLE 9 Rio Bravo Green Thunder True Heart 2.0 2.1 2.0 1.8 2.0 1.9 1.9 1.8 1.9 1.7 1.9 2.0 1.9 1.9 2.1 2.0 1.9 2.0 2.0 1.9 2.2 1.8 2.0 2.0 2.0 1.8 2.0 2.0 1.9 1.9 1.8 2.0 2.1 1.9 2.0 1.9 1.9 2.0 1.9 2.1 2.0 2.5 2.1 2.3 1.9 1.9 2.0 1.9 1.8 2.1 1.9 1.8 2.0 1.9 2.0 1.8 2.1 1.9 2.2 2.0 Average 1.9126 1.9662 1.9945

Tables 10 through 12 show the plant weight in grams at harvest maturity for Rio Bravo, (Table 10) Green Thunder, (Table 11) and True Heart (Table 12). The data were taken in 2008 TO 2010 at seven trials in WELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20 samples of each variety.

TABLE 10 Rio Bravo 1 2 3 4 5 6 7 354 659 379 870 1035 896 1040 425 644 487 875 860 683 940 380 669 606 868 743 596 990 308 597 525 870 896 759 990 401 493 613 1042 931 587 1020 370 640 568 949 687 703 1040 498 608 497 756 936 752 1080 471 606 605 703 870 817 1140 356 621 551 1043 1044 782 890 250 631 582 683 906 791 800 374 539 579 696 954 765 1240 436 589 506 1036 950 723 1270 319 651 411 798 945 594 1000 411 649 612 975 878 680 1140 364 653 501 872 829 692 1030 297 637 544 1014 911 746 1020 488 631 609 869 787 544 1090 316 610 596 742 931 763 950 361 612 554 827 931 583 1110 447 633 561 902 949 568 850 Sum 7626 12372 10886 17390 17973 14024 20630 Average 381.30 618.60 544.30 869.50 898.65 701.20 1031.50 Variance 4404.33 1743.94 4294.01 13456.37 7575.29 9217.75 13802.89

TABLE 11 Green Thunder 1 2 3 4 5 6 7 436 680 605 838 749 583 550 599 605 767 712 884 598 730 487 631 581 818 833 534 730 448 614 457 820 830 752 710 449 656 613 743 678 602 940 486 567 435 825 789 726 840 587 608 600 892 875 452 940 463 594 482 740 881 632 900 535 653 548 820 746 672 830 562 648 516 748 888 598 630 395 662 707 888 850 592 640 440 609 719 781 894 691 760 593 627 449 842 821 608 890 472 608 596 756 747 664 910 458 677 619 829 737 778 760 559 652 704 748 900 655 870 541 649 723 821 697 658 760 480 602 529 879 745 831 920 567 604 569 814 829 654 750 493 573 627 821 705 723 910 Sum 10050 12519 11846 16135 16078 13003 15970 Average 502.50 625.95 592.30 806.75 803.90 650.15 798.50 Variance 3651.11 1055.42 9477.91 2669.04 5328.83 7549.08 12918.68

TABLE 12 True Heart 1 2 3 4 5 6 7 495 850 588 795 732 1113 830 454 655 738 860 756 1003 990 451 671 704 775 777 981 830 521 769 792 707 858 969 800 645 743 522 672 810 958 740 484 586 740 745 783 993 1070 493 674 713 1012 728 947 890 472 698 664 850 622 935 870 546 584 900 794 709 1027 550 492 552 553 787 799 1116 880 342 739 492 795 955 944 870 552 774 893 588 850 811 920 474 692 702 875 682 898 850 481 637 740 714 781 901 850 441 798 527 694 745 888 760 628 579 787 891 751 891 980 566 598 593 779 638 981 830 481 688 711 687 771 828 920 433 682 746 832 659 1269 840 499 597 560 804 810 867 890 Sum 9950 13566 13665 15656 15216 19320 17160 Average 497.50 678.30 683.25 782.80 760.80 966.00 858.00 Variance 4575.21 6755.27 14006.0 8726.17 6240.06 11447.0 11174.74

Table 13 shows the results of a single-factor ANOVA testing showing differences in plant weight between varieties, locations, and interactions between variety and location for data in Tables 10-12. Column 1 shows the source of variation, column 2 shows the sum of squares, column 3 shows the degrees of freedom, column 4 shows the mean square, column 5 shows the F-value, column 6 shows the P-value, and column 7 shows the F critical value. ANOVA results indicate significant differences in plant weight between varieties at harvest maturity.

TABLE 13 De- Source grees of of Varia- Sum of Free- Mean tion Squares dom Square F-value P-value F crit Variety 288242.97 2 144121.4 18.9078 0.0000 3.02 Loca- 8417636.97 6 1402939.4 184.0562 0.0000 2.12 tion Inter- 2156705.26 12 179725.44 23.5788 0.0000 1.78 action Within 3041315.55 399 7622.34 Total 13903900.7 419

Tables 14 through 16 show the plant height in centimeters at harvest maturity for Rio Bravo, (Table 14) Green Thunder, (Table 15) and True Heart (Table 16). The data were taken in 2008 TO 2010 at seven trials in WELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20 samples of each variety.

TABLE 14 Rio Bravo 1 2 3 4 5 6 7 25 33 33 39 36 34 34 28 36 34 38 36 32 34 27 33 35 37 35 31 34 26 37 32 37 34 34 36 30 34 33 38 35 32 37 28 32 35 38 35 33 33 29 33 33 37 35 33 32 28 35 33 34 36 35 32 27 33 34 36 36 33 36 27 33 34 38 37 33 35 26 34 35 36 37 33 34 27 36 35 37 35 34 35 28 34 33 36 34 29 35 29 35 34 38 36 30 33 27 34 34 37 35 33 35 29 33 34 38 37 33 35 27 34 35 36 34 31 38 28 36 33 37 37 33 35 29 33 34 38 37 31 35 26 32 33 37 37 32 34 Sum 551 680 676 742 714 649 692 Average 27.55 34 33.8 37.1 35.7 32.45 34.6 Variance 1.6289 2.0000 0.8000 1.2526 1.1684 2.1553 2.2526

TABLE 15 Green Thunder 1 2 3 4 5 6 7 32 34 32 39 35 35 33 32 35 38 37 32 35 33 32 36 33 39 36 33 34 31 34 33 37 37 33 32 31 35 37 38 34 33 37 32 32 34 33 37 34 34 30 35 36 39 38 34 35 29 36 35 38 38 35 34 31 36 36 37 34 34 33 28 36 36 36 34 35 32 33 35 34 38 37 35 33 32 35 34 37 36 34 34 32 34 35 38 35 35 37 31 36 36 39 35 35 34 31 34 38 37 35 35 32 30 36 37 36 38 36 32 33 34 38 38 35 36 31 32 35 36 37 35 35 36 31 36 35 39 35 34 35 30 35 34 38 36 34 34 Sum 623 701 706 754 709 690 671 Average 31.15 35.05 35.30 37.70 35.45 34.50 33.55 Variance 1.6079 0.6816 3.2737 0.9579 2.4711 0.7895 3.4184

TABLE 16 True Heart 1 2 3 4 5 6 7 30 33 37 39 37 35 31 30 35 36 39 37 33 31 30 36 38 40 39 36 35 31 36 38 38 38 34 31 31 35 36 36 38 36 32 29 36 39 37 37 35 33 28 34 36 38 37 34 32 32 34 36 38 39 33 32 28 33 36 38 38 34 28 28 35 36 36 37 35 30 28 35 36 36 37 34 30 28 36 36 38 36 35 30 31 31 33 38 37 37 33 32 36 37 38 37 35 33 30 35 37 37 38 33 31 29 34 36 36 37 34 29 28 35 36 39 38 34 31 31 33 37 37 38 34 30 29 36 38 38 38 35 30 30 34 36 37 37 34 32 Sum 593 694 735 752 750 686 621 Average 29.65 34.70 36.75 37.60 37.50 34.30 31.05 Variance 1.9237 1.2737 0.9342 1.3053 0.5789 0.8526 2.4711

Table 17 shows the results of a single-factor ANOVA testing for plant height between varieties, and within the varieties, Rio Bravo, Green Thunder, and True Heart, for data in Tables 14-16. Column 1 shows the source of variation, column 2 shows the sum of squares, column 3 shows the degrees of freedom, column 4 shows the mean square, column 5 shows the F-value, column 6 shows the P-value, and column 7 shows the F critical value. ANOVA results indicate a significant difference in plant height between varieties at harvest maturity.

TABLE 17 De- grees of Source of Sum of Free Mean Variation Squares dom Square F-value P-value F crit Variety 93.2333 2 46.6167 28.9653 0.0000 3.0183 Location 2404.9952 6 400.8325 249.0574 0.0000 2.1213 Interaction 374.3333 12 31.1944 19.3827 0.0000 1.7765 Within 642.1500 399 1.6094 Total 3514.7119 419

Tables 18 through 20 show the frame length in centimeters at harvest maturity for Rio Bravo, (Table 18) Green Thunder, (Table 19) and True Heart (Table 20). The data were taken in 2008 TO 2010 at seven trials in WELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20 samples of each variety.

TABLE 18 Rio Bravo 1 2 3 4 5 6 7 26 29 31 34 32 31 33 27 31 30 35 34 30 32 26 32 31 33 32 29 33 26 31 29 35 34 30 34 24 31 31 33 36 29 30 26 30 29 34 33 29 34 24 29 31 35 32 29 32 27 31 30 33 34 30 34 26 32 29 33 34 31 33 25 31 29 34 34 29 33 27 31 31 33 33 31 32 26 30 30 35 33 28 32 26 32 30 35 33 28 29 25 30 31 34 35 28 27 26 30 29 33 35 30 29 26 31 30 34 34 29 31 25 32 31 34 34 29 33 27 31 30 35 34 30 31 27 28 31 33 30 29 31 26 30 31 35 32 27 33 Sum 518 612 604 680 668 586 636 Average 25.90 30.60 30.20 34.00 33.40 29.30 31.80 Variance 0.8316 1.2000 0.6947 0.7368 1.8316 1.1684 3.5368

TABLE 19 Green Thunder 1 2 3 4 5 6 7 27 32 32 31 35 30 31 28 32 33 33 34 32 30 29 30 32 32 35 33 32 28 31 29 33 36 30 29 29 32 34 34 35 33 32 27 27 33 33 30 35 32 26 30 33 34 35 32 29 28 32 32 33 34 31 30 26 32 31 33 34 32 30 27 33 33 33 36 31 28 27 33 33 32 36 32 31 26 31 29 34 34 31 31 27 32 31 31 36 31 33 28 31 31 32 35 32 30 29 32 32 34 36 31 30 26 33 33 31 34 33 29 27 31 33 33 34 32 27 28 32 30 33 35 33 31 29 32 31 34 36 32 33 29 30 32 32 35 34 32 Sum 551 634 637 652 700 637 607 Average 27.55 31.70 31.85 32.60 35.00 31.85 30.35 Variance 1.2079 0.9579 1.9237 1.4105 0.6316 1.0816 2.5553

TABLE 20 True Heart 1 2 3 4 5 6 7 27 30 35 34 36 31 26 26 31 35 36 35 32 27 25 30 35 36 37 32 26 26 32 36 35 37 31 25 27 30 34 33 36 31 28 28 31 33 36 36 31 28 26 30 35 34 36 32 26 27 31 35 36 35 32 27 26 30 36 36 35 34 26 25 32 36 35 37 31 31 27 31 34 35 35 31 28 28 30 34 35 36 33 30 25 25 28 36 36 37 31 28 31 34 34 37 33 28 26 30 35 36 37 33 29 27 31 35 33 36 33 28 26 30 35 35 37 32 29 26 30 33 35 36 33 30 26 30 34 34 36 33 31 27 31 34 34 37 30 30 Sum 529 609 694 698 724 639 561 Average 26.45 30.45 34.70 34.90 36.20 31.95 28.05 Variance 0.8921 0.7868 0.8526 1.0421 0.5895 1.1026 3.1026

Table 21 shows the results of a two-factor ANOVA testing for frame leaf length between varieties, and within the varieties, Rio Bravo, Green Thunder, and True Heart, for data in Tables 18-20. Column 1 shows the source of variation, column 2 shows the sum of squares, column 3 shows the degrees of freedom, column 4 shows the mean square, column 5 shows the F-value, column 6 shows the P-value, and column 7 shows the F critical value. ANOVA results indicate a significant difference in frame leaf length between varieties at harvest maturity.

TABLE 21 De- grees of Source of Sum of Free- Mean Variation Squares dom Square F-value P-value F crit Variety 87.6000 2 43.8000 32.6902 0.0000 3.0183 Location 2611.3571 6 435.2262 324.8321 0.0000 2.1213 Interaction 532.5000 12 44.3750 33.1194 0.0000 1.7765 Within 534.6000 399 1.3398 Total 3766.0571 419

Tables 22 through 24 shows the leaf width in centimeters at harvest maturity for Rio Bravo, (Table 22) Green Thunder, (Table 23) and True Heart (Table 24). The data were taken in 2008 TO 2010 at seven trials in WELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20 samples of each variety.

TABLE 22 Rio Bravo 1 2 3 4 5 6 7 17 19 18 19 22 24 24 18 21 16 21 19 21 21 16 20 17 21 21 22 24 16 19 18 21 22 22 23 16 20 18 21 23 20 21 16 20 18 20 20 19 25 15 19 17 19 22 21 21 17 19 18 20 19 21 27 16 20 17 21 22 21 24 16 19 18 20 22 22 24 17 20 17 21 22 21 24 18 19 18 20 21 17 23 16 20 16 21 21 19 24 17 21 17 20 22 20 24 16 18 18 21 20 21 23 16 18 18 21 20 21 22 18 19 18 19 20 19 23 16 20 17 19 22 20 22 18 19 18 20 19 20 23 16 19 17 20 20 17 21 Sum 331 389 349 405 419 408 463 Average 16.55 19.45 17.45 20.25 20.95 20.40 23.15 Variance 0.7868 0.6816 0.4711 0.6184 1.5237 2.7789 2.3447

TABLE 23 Green Thunder 1 2 3 4 5 6 7 18 19 19 19 17 20 19 17 20 18 20 18 18 20 18 19 18 18 18 21 21 17 19 16 17 17 21 19 17 20 20 18 18 20 21 17 16 18 19 19 18 18 16 20 19 19 17 19 19 17 18 18 19 17 17 22 17 19 18 18 17 22 20 18 21 20 17 17 18 18 18 19 17 17 17 20 17 17 20 17 18 18 20 20 16 18 19 18 18 18 20 18 18 18 19 17 21 23 18 19 19 20 17 20 22 17 19 18 19 17 19 22 16 19 18 17 17 21 21 17 20 19 19 18 22 23 17 20 19 18 18 19 19 18 19 17 18 18 20 20 Sum 343 384 366 367 349 394 406 Average 17.15 19.20 18.30 18.35 17.45 19.70 20.30 Variance 0.5553 0.6947 1.0632 0.8711 0.2605 2.0105 2.5368

TABLE 24 True Heart 1 2 3 4 5 6 7 16 19 20 19 16 19 18 18 19 21 21 16 21 20 17 19 19 19 18 22 19 17 18 18 20 17 19 20 16 19 23 20 17 20 18 17 18 23 23 18 19 21 18 18 19 21 17 19 18 17 19 22 20 16 20 20 16 19 18 20 18 19 20 16 19 17 19 17 20 21 18 20 17 22 16 20 18 17 19 19 20 18 20 19 17 17 19 21 20 17 19 16 19 21 21 17 21 20 17 18 23 20 18 18 21 16 18 18 19 18 21 18 17 19 17 19 18 18 18 18 18 17 20 18 19 20 17 19 19 22 17 21 20 16 20 20 21 17 20 21 Sum 337 376 392 406 344 395 390 Average 16.85 18.80 19.60 20.30 17.20 19.75 19.50 Variance 0.5553 0.3789 4.3579 1.2737 0.5895 1.1447 1.3158

Table 25 shows the results of a two-factor ANOVA testing for leaf width between varieties, and within the varieties, Rio Bravo, Green Thunder, and True Heart, for data in Tables 22-24. Column 1 shows the source of variation, column 2 shows the sum of squares, column 3 shows the degrees of freedom, column 4 shows the mean square, column 5 shows the F-value, column 6 shows the P-value, and column 7 shows the F critical value. ANOVA results indicate a significant difference in leaf width between varieties at harvest maturity.

Degrees Source of Sum of of Free- Mean Variation Squares dom Square F-value P-value F crit Variety 96.1000 2 48.0500 37.6326 0.0000 3.0183 Location 621.5571 6 103.5929 81.1337 0.0000 2.1213 Interaction 337.3000 12 28.1083 22.0144 0.0000 1.7765 Within 509.4500 399 1.2768 Total 1564.4071 419

Tables 26 through 28 show the leaf index (calculated by dividing the leaf length by the leaf width) at harvest maturity for Rio Bravo, (Table 26) Green Thunder, (Table 27) and True Heart (Table 28). The data were taken in 2008 TO 2010 at seven trials in WELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20 samples of each variety.

TABLE 26 Rio Bravo 1 2 3 4 5 6 7 1.53 1.53 1.72 1.79 1.45 1.29 1.38 1.50 1.48 1.88 1.67 1.79 1.43 1.52 1.63 1.60 1.82 1.57 1.52 1.32 1.38 1.63 1.63 1.61 1.67 1.55 1.36 1.48 1.50 1.55 1.72 1.57 1.57 1.45 1.43 1.63 1.50 1.61 1.70 1.65 1.53 1.36 1.60 1.53 1.82 1.84 1.45 1.38 1.52 1.59 1.63 1.67 1.65 1.79 1.43 1.26 1.63 1.60 1.71 1.57 1.55 1.48 1.38 1.56 1.63 1.61 1.70 1.55 1.32 1.38 1.59 1.55 1.82 1.57 1.50 1.48 1.33 1.44 1.58 1.67 1.75 1.57 1.65 1.39 1.63 1.60 1.88 1.67 1.57 1.47 1.21 1.47 1.43 1.82 1.70 1.59 1.40 1.13 1.63 1.67 1.61 1.57 1.75 1.43 1.26 1.63 1.72 1.67 1.62 1.70 1.38 1.41 1.39 1.68 1.72 1.79 1.70 1.53 1.43 1.69 1.55 1.76 1.84 1.55 1.50 1.41 1.50 1.47 1.72 1.65 1.58 1.45 1.35 1.63 1.58 1.82 1.75 1.60 1.59 1.57 Sum 31.3598 31.5068 34.6716 33.6393 31.9716 28.8533 27.5648 Aver- 1.5680 1.5753 1.7336 1.6820 1.5986 1.4427 1.3782 age Vari- 0.0060 0.0056 0.0082 0.0080 0.0100 0.0081 0.0117 ance

TABLE 27 Green Thunder 1 2 3 4 5 6 7 1.50 1.68 1.68 1.63 2.06 1.50 1.63 1.65 1.60 1.83 1.65 1.89 1.78 1.50 1.61 1.58 1.78 1.78 1.94 1.57 1.52 1.65 1.63 1.81 1.94 2.12 1.43 1.53 1.71 1.60 1.70 1.89 1.94 1.65 1.52 1.69 1.69 1.83 1.74 1.58 1.94 1.78 1.63 1.50 1.74 1.79 2.06 1.68 1.53 1.65 1.78 1.78 1.74 2.00 1.82 1.36 1.53 1.68 1.72 1.83 2.00 1.45 1.50 1.50 1.57 1.65 1.94 2.12 1.72 1.56 1.50 1.74 1.94 1.88 2.12 1.60 1.82 1.53 1.55 1.71 1.89 1.89 1.55 1.55 1.69 1.78 1.63 1.72 2.00 1.72 1.65 1.56 1.72 1.72 1.68 2.06 1.52 1.30 1.61 1.68 1.68 1.70 2.12 1.55 1.36 1.53 1.74 1.83 1.63 2.00 1.74 1.32 1.69 1.63 1.83 1.94 2.00 1.52 1.29 1.65 1.60 1.58 1.74 1.94 1.50 1.35 1.71 1.60 1.63 1.89 2.00 1.68 1.74 1.61 1.58 1.88 1.78 1.94 1.70 1.60 Sum 32.1646 33.0799 34.8761 35.6231 40.1471 32.4810 30.0811 Aver- 1.6082 1.6540 1.7438 1.7812 2.0074 1.6240 1.5041 age Vari- 0.0053 0.0079 0.0085 0.0132 0.0055 0.0139 0.0210 ance

TABLE 28 True Heart 1 2 3 4 5 6 7 1.69 1.58 1.75 1.79 2.25 1.63 1.44 1.44 1.63 1.67 1.71 2.19 1.52 1.35 1.47 1.58 1.84 1.89 2.06 1.45 1.37 1.53 1.78 2.00 1.75 2.18 1.63 1.25 1.69 1.58 1.48 1.65 2.12 1.55 1.56 1.65 1.72 1.43 1.57 2.00 1.63 1.33 1.44 1.67 1.84 1.62 2.12 1.68 1.44 1.59 1.63 1.59 1.80 2.19 1.60 1.35 1.63 1.58 2.00 1.80 1.94 1.79 1.30 1.56 1.68 2.12 1.84 2.18 1.55 1.48 1.50 1.55 2.00 1.59 2.19 1.55 1.56 1.65 1.58 1.79 1.75 2.00 1.65 1.58 1.47 1.47 1.47 1.71 1.80 2.18 1.63 1.75 1.63 1.62 1.62 2.18 1.57 1.40 1.53 1.67 1.52 1.80 2.06 1.83 1.38 1.69 1.72 1.94 1.74 2.00 1.57 1.56 1.53 1.58 2.06 1.84 2.06 1.78 1.61 1.44 1.67 1.94 1.75 2.00 1.74 1.50 1.53 1.58 1.79 1.55 2.12 1.57 1.55 1.69 1.55 1.70 1.62 2.18 1.50 1.43 Sum 31.4620 32.4275 35.8009 34.4783 42.1589 32.4406 28.8331 Aver- 1.5731 1.6214 1.7900 1.7239 2.1079 1.6220 1.4417 age Vari- 0.0095 0.0053 0.0399 0.0105 0.0076 0.0101 0.0106 ance

Table 29 shows the results of a single-factor ANOVA testing for the leaf index between varieties, and within the varieties, Rio Bravo, Green Thunder, and True Heart, for data in Tables 26-28. Column 1 shows the source of variation, column 2 shows the sum of squares, column 3 shows the degrees of freedom, column 4 shows the mean square, column 5 shows the F-value, column 6 shows the P-value, and column 7 shows the F critical value. ANOVA results indicate a significant difference in the leaf index between varieties at harvest maturity.

TABLE 29 Source of Sum of Degrees of Mean Variation Squares Freedom Square F-value P-value F crit Variety 1.6253 2 0.8126 75.3459 0.0000 3.0183 Location 8.3222 6 1.3870 128.6010 0.0000 2.1213 Interaction 2.0950 12 0.1746 16.1867 0.0000 1.7765 Within 4.3034 399 0.0108 Total 16.3460 419

Tables 30 through 32 show the leaf area (calculated by multiplying the leaf length by the leaf width) at harvest maturity for Rio Bravo, (Table 30) Green Thunder, (Table 31) and True Heart (Table 32). The data were taken in 2008 TO 2010 at seven trials in WELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20 samples of each variety.

TABLE 30 Rio Bravo 1 2 3 4 5 6 7 442 551 558 646 704 744 792 486 651 480 735 646 630 672 416 640 527 693 672 638 792 416 589 522 735 748 660 782 384 620 558 693 828 580 630 416 600 522 680 660 551 850 360 551 527 665 704 609 672 459 589 540 660 646 630 918 416 640 493 693 748 651 792 400 589 522 680 748 638 792 459 620 527 693 726 651 768 468 570 540 700 693 476 736 416 640 480 735 693 532 696 425 630 527 680 770 560 648 416 540 522 693 700 630 667 416 558 540 714 680 609 682 450 608 558 646 680 551 759 432 620 510 665 748 600 682 486 532 558 660 570 580 713 416 570 527 700 640 459 693 Sum 8579 11908 10538 13766 14004 11979 14736 Average 428.95 595.40 526.90 688.30 700.20 598.95 736.80 Variance 1022.78 1373.41 542.83 740.01 3201.12 4334.16 5477.01

TABLE 31 Green Thunder 1 2 3 4 5 6 7 486 608 608 589 595 600 589 476 640 594 660 612 576 600 522 570 576 576 630 693 672 476 589 464 561 612 630 551 493 640 680 612 630 660 672 432 432 594 627 570 630 576 416 600 627 646 595 608 551 476 576 576 627 578 527 660 442 608 558 594 578 704 600 486 693 660 561 612 558 504 486 627 561 544 612 640 527 442 620 493 612 612 620 620 432 576 589 558 648 558 660 504 558 558 608 595 672 690 522 608 608 680 612 620 660 442 627 594 589 578 627 638 432 589 594 561 578 672 567 476 640 570 627 630 726 713 493 640 589 612 648 608 627 522 570 544 576 630 680 640 Sum 9456 12173 11670 11963 12215 12555 12321 Average 472.80 608.60 583.50 598.15 610.75 627.75 616.05 Variance 1088.48 1084.03 2456.47 1357.61 509.04 2867.04 3263.94

TABLE 32 True Heart 1 2 3 4 5 6 7 432 570 700 646 576 589 468 468 589 735 756 560 672 540 425 570 665 684 666 704 494 442 576 648 700 629 589 500 432 570 782 660 612 620 504 476 558 759 828 648 589 588 468 540 665 714 612 608 468 459 589 770 720 560 640 540 416 570 648 720 630 646 520 400 608 612 665 629 620 651 486 620 578 770 560 620 504 476 570 646 700 648 660 570 425 425 532 756 720 629 589 448 589 714 714 629 693 560 442 540 805 720 666 594 609 432 558 630 627 648 693 504 442 570 595 665 666 576 522 468 540 561 700 648 627 600 442 570 646 748 612 693 620 432 620 680 714 629 600 630 Sum 8911 11449 13595 14171 12457 12622 10952 Average 445.55 572.45 679.75 708.5500 622.85 631.10 547.60 Variance 514.05 624.99 4971.04 2105.31 1197.61 1737.25 2985.62

Table 33 shows the results of a two-factor ANOVA testing for the leaf area between varieties, and within the varieties, Rio Bravo, Green Thunder, and True Heart, for data in Tables 30-32. Column 1 shows the source of variation, column 2 shows the sum of squares, column 3 shows the degrees of freedom, column 4 shows the mean square, column 5 shows the F-value, column 6 shows the P-value, and column 7 shows the F critical value. ANOVA results indicate a significant difference in leaf area between varieties at harvest maturity.

TABLE 33 Source of Sum of Degrees of Mean Variation Squares Freedom Square F-value  P-value F crit Variety 35837.3190 2 17918.659 8.6596 0.0002 3.0183 Location 1832966.014 6 305494.34 147.6368 0.0000 2.1213 Interaction 848011.1143 12 70667.593 34.1516 0.0000 1.7765 Within 825622.6000 399 2069.2296 Total 3542437.047 419

Tables 34 through 36 show the core length in centimeters at harvest maturity for Rio Bravo, (Table 34) Green Thunder, (Table 35) and True Heart (Table 36). The data were taken in 2008 TO 2010 at seven trials in WELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20 samples of each variety.

TABLE 34 Rio Bravo 1 2 3 4 5 6 7 3.8 5.6 6.0 9.0 5.9 8.0 8.1 3.2 6.1 6.0 8.3 6.9 6.8 8.1 4.0 5.2 4.3 7.9 6.0 7.1 7.0 3.6 5.4 5.4 8.5 5.4 7.9 10.0 3.5 5.7 5.7 7.9 5.4 6.2 8.0 3.2 6.0 6.0 7.9 5.2 7.3 8.2 3.5 6.0 4.8 8.7 6.2 9.0 9.1 3.6 5.9 5.6 8.1 7.1 9.0 8.2 4.0 5.8 6.1 8.3 6.5 9.0 8.6 3.9 6.1 5.9 8.8 6.6 9.0 9.2 3.4 5.5 5.3 7.9 7.1 8.0 8.1 3.3 5.6 5.4 8.2 6.4 9.0 6.0 3.7 5.9 5.3 8.4 6.0 6.0 7.2 3.7 6.1 5.9 8.1 7.0 7.0 8.0 3.8 6.0 5.0 8.6 4.5 6.5 8.1 4.0 5.6 5.6 8.6 6.8 7.0 7.5 3.8 6.0 5.9 8.4 5.9 6.0 7.0 3.5 6.3 6.1 8.1 6.8 7.0 8.4 3.6 6.0 5.4 8.3 7.5 6.1 8.5 3.4 5.8 4.9 8.4 7.1 5.6 8.4 Sum 72.5 116.6 110.6 166.4 126.3 147.5 161.7 Average 3.6250 5.8300 5.5300 8.3200 6.3150 7.3750 8.0850 Variance 0.0641 0.0769 0.2443 0.1006 0.6013 1.3578 0.7603

TABLE 35 Green Thunder 1 2 3 4 5 6 7 4.4 5.0 5.5 9.1 6.0 6.7 6.0 4.1 5.8 5.6 8.2 4.8 7.0 7.5 3.7 4.9 3.6 8.8 5.9 6.5 7.5 3.7 5.9 3.2 7.9 6.1 7.7 6.5 3.4 5.2 5.2 8.2 6.2 7.5 8.5 4.2 4.2 5.2 5.2 6.0 5.3 8.5 4.2 5.5 5.3 8.4 5.4 7.2 8.0 3.6 4.9 4.5 8.6 6.0 7.5 8.5 4.5 5.7 5.1 8.1 5.0 7.0 7.5 3.3 5.1 5.8 6.4 6.1 6.5 6.0 3.6 5.3 5.6 7.7 6.0 8.0 7.0 3.2 5.8 4.1 8.8 4.6 8.0 9.0 4.2 4.9 4.3 8.9 5.9 6.0 6.0 4.6 6.0 3.8 7.8 6.0 7.0 6.3 4.1 6.2 4.1 8.1 5.7 8.0 6.5 3.2 5.8 5.7 6.9 5.0 6.6 6.7 4.0 5.7 5.2 8.7 4.6 7.0 7.7 4.3 5.0 5.3 8.0 5.8 6.0 8.0 4.3 5.8 4.9 7.9 5.1 6.2 8.5 3.7 5.3 5.5 8.4 5.9 6.9 7.5 Sum 78.3 109 97.5 160.9 111.4 141.8 145.2 Average 3.9150 5.4500 4.8750 8.0450 5.5700 7.0900 7.2600 Variance 0.1929 0.1732 0.5978 0.6552 0.2917 0.5009 0.9457

TABLE 36 True Heart 1 2 3 4 5 6 7 3.2 6.1 5.1 6.7 5.5 8.2 6.5 3.6 5.7 5.7 7.2 5.7 6.2 7.8 3.6 6.1 5.6 6.3 5.6 7.1 7.2 3.9 6.8 5.2 7.7 6.3 7.0 5.0 4.7 4.6 5.0 7.1 6.7 7.1 5.7 4.0 5.4 4.9 6.1 5.4 5.9 7.3 4.8 6.7 5.1 7.5 5.8 7.2 8.0 3.1 6.1 5.6 7.1 5.7 6.2 8.0 3.5 6.0 5.5 6.5 5.5 6.1 5.8 3.8 6.4 5.1 6.8 5.3 5.6 6.0 4.0 5.6 5.6 6.0 5.5 6.4 6.8 3.5 6.3 5.3 7.2 5.2 6.5 5.2 3.8 3.8 6.2 5.3 7.0 5.0 7.0 3.2 6.1 5.4 6.8 6.2 5.2 6.3 4.6 5.6 5.6 6.7 6.1 5.8 8.5 4.4 4.9 4.9 6.3 5.4 7.9 8.0 3.6 6.1 5.1 6.7 5.5 6.2 8.2 3.3 5.6 5.3 7.2 6.4 6.6 7.2 3.8 5.9 5.7 7.7 5.4 6.4 6.7 4.4 6.7 5.7 6.9 5.5 5.8 7.6 Sum 76.8 118.9 106.7 137.5 113.7 130.4 138.5 Average 3.8400 5.9450 5.3350 6.8750 5.6850 6.5200 6.9250 Variance 0.2625 0.3163 0.0761 0.2346 0.1919 0.5712 1.0567

Table 37 shows the results of a two-factor ANOVA testing for the core length between varieties, and within the varieties, Rio Bravo, Green Thunder, and True Heart, for data in Tables 34-36. Column 1 shows the source of variation, column 2 shows the sum of squares, column 3 shows the degrees of freedom, column 4 shows the mean square, column 5 shows the F-value, column 6 shows the P-value, and column 7 shows the F critical value. ANOVA results indicate a significant difference in core length between varieties at harvest maturity.

TABLE 37 De- grees of Source of Sum of Free- Mean Variation Squares dom Square F-value P-value F crit Variety 23.8800 2 11.9400 27.0428 0.0000 3.0183 Location 689.9235 6 114.9872 260.4327 0.0000 2.1213 Interaction 36.0570 12 3.0047 6.8054 0.0000 1.7765 Within 176.1680 399 0.4415 Total 926.0285 419

Tables 38 through 40 show the core diameter in centimeters at harvest maturity for Rio Bravo, (Table 38) Green Thunder, (Table 39) and True Heart (Table 40). The data were taken in 2008 TO 2010 at seven trials in WELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20 samples of each variety.

TABLE 38 Rio Bravo 1 2 3 4 5 6 7 3.0 3.2 3.2 3.8 3.5 3.5 4.0 2.8 3.0 3.2 3.3 3.8 3.4 4.0 3.0 3.0 3.0 4.0 3.5 3.3 4.1 2.7 3.1 3.1 4.0 3.5 3.5 3.8 3.0 3.3 3.0 3.5 3.8 3.2 3.9 3.0 3.2 3.0 3.5 3.9 3.5 4.5 3.0 3.0 3.1 3.8 4.0 3.5 4.4 2.9 3.0 3.2 3.9 3.5 3.8 3.8 3.0 3.2 3.0 3.4 4.0 3.9 4.2 3.1 3.1 3.1 3.6 3.7 3.9 3.7 3.0 3.1 3.0 3.7 3.7 3.9 4.3 2.9 3.3 3.1 4.0 3.8 3.5 4.2 3.1 3.2 3.1 3.7 3.7 3.6 5.2 3.0 3.1 3.2 3.9 3.6 3.5 4.3 3.2 3.0 3.0 3.9 3.8 4.0 4.4 3.2 3.2 3.1 3.7 3.7 4.0 4.5 3.4 3.1 3.2 3.9 3.9 3.7 4.0 2.9 3.0 3.1 4.0 3.9 3.9 4.1 2.8 3.3 3.0 3.8 3.9 3.5 4.3 2.8 3.1 3.3 3.8 3.9 3.5 4.0 Sum 59.8 62.5 62 75.2 75.1 72.6 83.7 Aver- 2.9900 3.1250 3.1000 3.7600 3.7550 3.6300 4.1850 age Vari- 0.0262 0.0114 0.0084 0.0436 0.0279 0.0569 0.1119 ance

TABLE 39 Green Thunder 1 2 3 4 5 6 7 3.1 3.6 4.0 3.8 3.2 3.7 3.5 3.3 3.4 3.0 4.0 3.3 3.5 4.0 3.4 3.2 3.5 4.0 3.8 3.5 4.0 3.5 3.4 3.0 3.9 3.2 3.8 4.3 3.3 3.7 3.4 4.1 3.5 3.8 4.5 3.0 3.0 3.7 3.0 3.9 3.6 3.8 3.6 3.8 3.9 4.0 3.7 3.8 4.0 3.0 3.3 3.5 4.1 3.6 3.8 4.5 3.2 3.8 3.6 4.1 3.8 3.8 4.3 3.4 3.3 3.2 3.8 3.6 3.4 4.3 3.3 3.5 3.2 3.8 3.6 3.8 4.0 3.0 3.6 3.8 3.8 3.5 3.9 4.5 3.1 3.3 3.6 4.0 3.5 3.5 4.5 3.6 3.7 3.5 3.9 3.8 3.5 5.0 3.2 3.2 3.4 3.9 3.5 3.5 4.4 3.1 3.5 3.6 3.8 3.5 3.7 4.3 3.4 3.6 3.6 4.0 3.5 3.5 4.5 3.7 3.4 3.7 4.0 3.5 3.2 4.3 3.2 3.3 3.8 3.8 3.2 3.6 4.2 3.4 3.8 3.5 3.9 3.2 3.7 4.0 Sum 65.8 70.1 69.8 78.6 70.1 72.8 85.6 Aver- 3.2900 3.5050 3.4900 3.9300 3.5050 3.6400 4.2800 age Vari- 0.0441 0.0416 0.0852 0.0117 0.0394 0.0331 0.0964 ance

TABLE 40 True Heart 1 2 3 4 5 6 7 3.4 3.6 4.0 3.6 3.2 3.8 4.5 3.3 3.8 3.9 4.0 3.3 3.5 4.2 3.2 3.5 3.9 3.8 3.8 3.6 4.0 3.3 3.9 3.8 3.9 3.2 3.8 4.0 3.5 3.6 3.6 3.8 3.5 4.0 4.0 3.5 3.2 3.3 4.0 3.6 3.1 4.0 3.2 3.9 3.6 3.8 3.7 3.7 4.5 3.0 3.8 3.9 4.0 3.6 3.4 4.0 3.2 3.8 3.4 3.9 3.8 3.1 4.1 3.4 3.7 3.8 3.8 3.6 3.6 4.0 3.1 3.4 3.8 3.6 3.6 3.7 4.1 3.2 3.7 4.0 3.7 3.5 3.8 4.2 3.5 3.5 3.5 3.8 4.0 3.5 3.7 3.4 3.6 3.8 4.0 3.8 3.6 4.2 3.3 3.8 3.6 3.7 3.5 3.7 4.5 3.3 3.6 3.7 3.8 3.5 3.7 4.8 3.5 3.7 3.5 3.8 3.5 3.7 4.0 3.4 3.3 3.7 3.9 3.5 3.8 4.6 3.3 3.9 3.8 4.0 3.2 3.8 4.5 3.4 4.0 3.8 3.9 3.2 3.6 4.5 Sum 66.4 73.3 74.7 77 70.1 72.7 84.9 Aver- 3.3200 3.6650 3.7350 3.8500 3.5050 3.6350 4.2450 age Vari- 0.0196 0.0445 0.0350 0.0174 0.0394 0.0498 0.0647 ance

Table 41 shows the results of a single-factor ANOVA testing for the core diameter between varieties, and within the varieties, Rio Bravo, Green Thunder, and True Heart, for data in Tables 38-40. Column 1 shows the source of variation, column 2 shows the sum of squares, column 3 shows the degrees of freedom, column 4 shows the mean square, column 5 shows the F-value, column 6 shows the P-value, and column 7 shows the F critical value. ANOVA results indicate a significant difference in core diameter between varieties at harvest maturity.

TABLE 41 Source of Sum of Degrees of Mean Variation Squares Freedom Square F-value P-value F crit Variety 3.1299 2 1.5649 36.1870 0.0000 3.0183 Location 40.5796 6 6.7633 156.3920 0.0000 2.1213 Interaction 6.5978 12 0.5498 12.7138 0.0000 1.7765 Within 17.2550 399 0.0432 Total 67.5623 419

DEPOSIT INFORMATION

A deposit of the lettuce cultivar seed of this invention is maintained by Syngenta Participations A.G, Inc., having an address at Werk Rosental, Schwarzwaldallee 215, CH-4058 Basel. A deposit of the cultivar Rio Bravo has been made with the American Type Culture Collection (ATCC), Manassas, Va. 20110, ATCC Deposit No.: PTA-11834.

Access to this deposit will be available during the pendency of this application to persons determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. Upon allowance of any claims in this application, all restrictions on the availability to the public of the variety will be irrevocably removed by affording access to a deposit of at least 2,500 seeds of the same variety with the American Type Culture Collection, Manassas, Va.

While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions, and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions, and sub-combinations as are within their true spirit and scope. 

What is claimed is:
 1. A seed of lettuce cultivar Rio Bravo, wherein a representative sample of seed of said cultivar was deposited under ATCC Accession No. PTA-11834.
 2. A lettuce plant, or a part thereof, produced by growing the seed of claim
 1. 3. A tissue culture produced from protoplasts or cells from the plant of claim 2, wherein said cells or protoplasts are produced from a plant part selected from the group consisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell root, root tip, pistil, anther, ovule, flower, shoot, stem, seed, and petiole.
 4. A lettuce plant regenerated from the tissue culture of claim 3, wherein the plant has all of the morphological and physiological characteristics of cultivar Rio Bravo.
 5. A method for producing a lettuce seed comprising crossing two lettuce plants and harvesting the resultant lettuce seed, wherein at least one lettuce plant is the lettuce plant of claim
 2. 6. A lettuce seed produced by the method of claim
 5. 7. A lettuce plant, or a part thereof, produced by growing said seed of claim
 6. 8. The method of claim 5, wherein at least one of said lettuce plants is transgenic.
 9. A method of producing a male sterile lettuce plant, wherein the method comprises introducing a nucleic acid molecule that confers male sterility into the lettuce plant of claim
 2. 10. A male sterile lettuce plant produced by the method of claim
 9. 11. A method of producing an herbicide resistant lettuce plant, wherein said method comprises introducing a gene conferring herbicide resistance into the plant of claim 2, wherein the gene is selected from the group consisting of glyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxy proprionic acid, L-phosphinothricin, cyclohexone, cyclohexanedione, triazine, and benzonitrile.
 12. An herbicide resistant lettuce plant produced by the method of claim
 11. 13. A method of producing a pest or insect resistant lettuce plant, wherein said method comprises introducing a gene conferring pest or insect resistance into the plant of claim
 2. 14. A pest or insect resistant lettuce plant produced by the method of claim
 13. 15. The lettuce plant of claim 14, wherein the gene encodes a Bacillus thuringiensis endotoxin.
 16. A method of producing a disease resistant lettuce plant, wherein said method comprises introducing a gene conferring disease resistance into the plant of claim
 2. 17. A disease resistant lettuce plant produced by the method of claim
 16. 18. A method of producing a lettuce plant with a value-added trait, wherein said method comprises introducing a gene conferring a value-added trait into the plant of claim 2, where said gene encodes a protein selected from the group consisting of a ferritin, a nitrate reductase, and a monellin.
 19. A lettuce plant with a value-added trait produced by the method of claim
 18. 20. A method of introducing a desired trait into lettuce cultivar Rio Bravo wherein the method comprises: (a) crossing a Rio Bravo plant, wherein a representative sample of seed was deposited under ATCC Accession No. PTA-11834, with a plant of another lettuce cultivar that comprises a desired trait to produce progeny plants wherein the desired trait is selected from the group consisting of male sterility, herbicide resistance, insect or pest resistance, modified bolting and resistance to bacterial disease, fungal disease or viral disease; (b) selecting one or more progeny plants that have the desired trait to produce selected progeny plants; (c) crossing the selected progeny plants with the Rio Bravo plant to produce backcross progeny plants; (d) selecting for backcross progeny plants that have the desired trait and all of the physiological and morphological characteristics of lettuce cultivar Rio Bravo listed in Table 1; and (e) repeating steps (c) and (d) two or more times in succession to produce selected third or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of lettuce cultivar Rio Bravo listed in Table
 1. 21. A lettuce plant produced by the method of claim 20, wherein the plant has the desired trait.
 22. The lettuce plant of claim 21, wherein the desired trait is herbicide resistance and the resistance is conferred to an herbicide selected from the group consisting of glyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxy proprionic acid, L-phosphinothricin, cyclohexone, cyclohexanedione, triazine, and benzonitrile.
 23. The lettuce plant of claim 21, wherein the desired trait is insect or pest resistance and the insect or pest resistance is conferred by a transgene encoding a Bacillus thuringiensis endotoxin. 